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Multi-epitope peptide-loaded DC (dendritic cell) therapeutic vaccine for HCV (hepatitis C viruses)

A hepatitis C virus and dendritic cell technology, applied in the fields of virology and immunology, can solve the problems of lack of protective antibodies, insufficient understanding of pathogenesis, and inability to prevent patients from reinfection

Inactive Publication Date: 2011-10-12
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Due to the necessity and urgency of HCV vaccine research, since the HCV gene was cloned in 1989, people have been working on vaccine research, but the progress is slow.
Earlier, people focused on the development of HCV envelope glycoprotein or polypeptide subunit vaccines. Like HIV vaccine research, due to the highly variable nature of HCV envelope glycoproteins, it is possible to produce neutralizing antibodies to control virus infection and replication. Encounter difficulties
The main reasons why the HCV vaccine is difficult to achieve are summarized in the following three points: ① The HCV genome has a high mutation rate, multiple virus quasi-species, lack of protective antibodies, and cannot prevent reinfection of chimpanzee and human recovered patients. Traditional preventive vaccine research has encountered difficulties. ② HCV has poor replication ability and weak infectivity, which makes the virus titer in the body low, and it is not easy to cultivate in vitro. In addition, the understanding of the virus replication infection process and pathogenesis is not clear enough, and many proteins encoded by HCV, such as The core protein and NS protein may also be related to the carcinogenicity of HCV, and the selection and strength of the immunogen are limited to a certain extent; ③ There is a lack of ideal in vitro infection cell models, and animal models except chimpanzees, other animals, even primates, For example, baboons and rhesus monkeys cannot be infected by HCV, which limits the research on vaccine evaluation

Method used

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  • Multi-epitope peptide-loaded DC (dendritic cell) therapeutic vaccine for HCV (hepatitis C viruses)
  • Multi-epitope peptide-loaded DC (dendritic cell) therapeutic vaccine for HCV (hepatitis C viruses)
  • Multi-epitope peptide-loaded DC (dendritic cell) therapeutic vaccine for HCV (hepatitis C viruses)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1. Expression of recombinant adenovirus:

[0028]1. Construction of recombinant adenovirus expression plasmids AD-sequence 1 and AD-sequence 2: Sequence 1 in pGH and Sequence 2 in pGH were synthesized by Generay Biotech (Shanghai). Sequence 1 is HCV CTL epitope NS4B (1793-1801) SMMAFSAAL and P7 (774-782) AAWYIKGRL plus HCV Th epitope NS3 (1248-1261) GYKVLVLNPSVAAT in series, the middle of the epitope is connected by AAY that promotes the presentation of the epitope , using both pichia yeast biased codons and Escherichia coli biased codons to design genes, and artificially synthesize HCV CTL bi-epitope gene tandem. Sequence 2 is a positive control, including HCV CTL epitope Core(35-44)YLLPRRGPRL, Core(132-140)DLMGYIPLV and HCV Th epitope NS3(1248-1261)GYKVLVLNPSVAAT. Sequences 1 and 2 are added with GFP and FLAG tags at the same time, which is beneficial to Western Blot detection of the expression of the target polypeptide. Double digestion with restriction end...

Embodiment 2

[0030] Example 2. Culture of dendritic cells

[0031] 1. Cultivate dendritic cells: Whole blood comes from healthy donors. Peripheral blood mononuclear cells (PBMC) are separated by Ficoll-Hypaque density gradient centrifugation. CD14+ mononuclear cells are separated and collected according to the CD14 MicroBeads humanmonocyte kit (MACS) operating instructions. cells, the cell purity was 96.32%. Adjust the density of CD14+ monocytes to 1×10 with serum-free medium X-VIVO15 6 Inoculate cells / mL in 24-well plate, incubate at 37°C in 5% CO2 for 5 days, change half of the medium every other day, and add GM-CSF (1000U / ml) and IL-4 (1000U / ml) from the 5th day Mature dendrites can be induced by maturation-inducing factors rTNF-α (10ng / mL), IL-1β (10 ng / mL), IL-6 (10 ng / mL), and PGE2 (1μg / mL) for 2 days shaped cells. Observed under an inverted microscope, after 1 day of culture, most of the cells still adhered to the wall and arranged in clusters, round in shape, with smooth cell me...

Embodiment 3

[0036] Example 3. Detection of Immunogenicity of Dendritic Cell Vaccines

[0037] 1. Mixed lymphocyte culture: DCs infected with recombinant adenovirus and not infected with recombinant adenovirus were collected as stimulator cells. Stimulate cells at 1×10 3 / hole, 5×10 3 / hole, 1×10 4 Each well was inoculated in a 96-well culture plate, and each group had three replicate wells; CD14-PBMC were used as effector cells, and 1×10 5 At the same time, only the effector cells were added as the negative control, and only the culture medium was added as the blank control, and the final volume of each well was 200 μl. The mixed lymphocytes were cultured under the conditions of 5% CO2 and 37°C. After 4 days, 20 μl / well of MTS solution was added, and the incubation was continued for 4 hours. The enzyme-linked immunoassay analyzer detected the A450 value at a wavelength of 450 nm, and the results were expressed as the average value of 3 wells. Stimulation index (SI)=experimental group-...

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Abstract

The invention discloses a multi-epitope peptide-loaded DC (dendritic cell) therapeutic vaccine for HCV (hepatitis C viruses), relating to the technical field of virology and immunology. The multi-epitope peptide-loaded DC (dendritic cell) therapeutic vaccine is characterized in that two CTL epitopes (namely, a NS4B (1793-1801) SMMAFSAAL and a P7 (774-782) AAWYIKGRL) are used for constructing recombinant adenoviruses, then the recombinant adenoviruses are used for infecting human dendritic cells so as to prepare a multi-epitope DC vaccine. Detection results indicate that the multi-epitope peptide-loaded DC (dendritic cell) therapeutic vaccine for HCV (hepatitis C viruses) disclosed by the invention has an immunogenicity.

Description

technical field [0001] The invention relates to virology and immunology techniques, and constructs a dendritic cell therapeutic vaccine loaded with multi-epitope peptides of hepatitis C virus. Background technique [0002] Hepatitis C virus (HCV) is one of the main pathogens that cause chronic liver disease. There are about 170 million to 200 million HCV infected people in the world. The HCV infection rate of the Chinese population is about 3.2%. It is estimated that there are more than 40 million infected people nationwide. After HCV infection, about 50% -85% turn into chronic hepatitis, of which 20% -30% develop into liver cirrhosis, and some turn into hepatocellular carcinoma (HCC), which seriously endangers human health and has become a global and severe public health problem. Hygiene issue. So far, there is still a lack of ideal antiviral treatment measures, and interferon treatment is not satisfactory. Due to the high mutation rate of HCV virus, the research of preve...

Claims

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Application Information

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IPC IPC(8): A61K39/39G01N33/576G01N33/569C12N15/861A61P1/16A61P31/14
Inventor 贾战生周云潘蕾李端
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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